Chemical modification of proteins to improve biocompatibility and bioactivity

ABSTRACT

The present invention broadly relates to chemical modification of biologically active proteins or analogs thereof. More specifically, the present invention describes novel methods for site-specific chemical modification of various proteins, and resultant compositions having improved biocompatibility and bioactivity.

[0001] This application is a division of application Ser. No.09/422,396, filed Oct. 21, 1999, which is a divisional of applicationSer. No. 09/119,800, filed Jul. 21, 1998, granted Pat. No. 6,017,876,which is a CIP of application Ser. No. 08/911,224, filed Aug. 15, 1997,granted Pat. No. 5,900,404, which are hereby incorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention broadly relates to chemical modification ofbiologically active proteins or analogs thereof (the term “protein” asused herein is synonymous with “polypeptide” or “peptide” unlessotherwise indicated). More specifically, the present invention describesnovel methods for site-specific chemical modifications of variousproteins, and resultant compositions.

BACKGROUND OF THE INVENTION

[0003] Due to recent advances in genetic and cell engineeringtechnologies, proteins known to exhibit various pharmacological actionsin vivo are capable of production in large amounts for pharmaceuticalapplications. Such proteins include erythropoietin (EPO), granulocytecolony-stimulating factor (G-CSF), interferons (alpha, beta, gamma,consensus), tumor necrosis factor binding protein (TNFbp), interleukin-1receptor antagonist (IL-1ra), brain-derived neurotrophic factor (BDNF),kerantinocyte growth factor (KGF), stem cell factor (SCF), megakaryocytegrowth differentiation factor (MGDF), osteoprotegerin (OPG), glial cellline derived neurotrophic factor (GDNF) and obesity protein (OBprotein). OB protein may also be referred to herein as leptin.

[0004] Leptin is active in vivo in both ob/ob mutant mice (mice obesedue to a defect in the production of the OB gene product) as well as innormal, wild type mice. The biological activity manifests itself in,among other things, weight loss. See generally, Barinaga, “Obese”Protein Slims Mice, Science 269: 475-476 (1995) and Friedman, “TheAlphabet of Weight Control,” Nature 385: 119-120 (1997). It is known,for instance, that in ob/ob mutant mice, administration of leptinresults in a decrease in serum insulin levels, and serum glucose levels.It is also known that administration of leptin results in a decrease inbody fat. This was observed in both ob/ob mutant mice, as well asnon-obese normal mice. Pelleymounter et al., Science 269: 540-543(1995); Halaas et al., Science 269: 543-546 (1995). See also, Campfieldet al., Science 269: 546-549 (1995) (Peripheral and centraladministration of microgram doses of leptin reduced food intake and bodyweight of ob/ob and diet-induced obese mice but not in db/db obesemice.) In none of these reports have toxicities been observed, even atthe highest doses.

[0005] Preliminary leptin induced weight loss experiments in animalmodels predict the need for a high concentration leptin formulation withchronic administration to effectively treat human obesity. Dosages inthe milligram protein per kilogram body weight range, such as 0.5 or 1.0mg/kg/day or below, are desirable for injection of therapeuticallyeffective amounts into larger mammals, such as humans. An increase inprotein concentration is thus necessary to avoid injection of largevolumes, which can be uncomfortable or possibly painful to the patient.

[0006] Unfortunately, for preparation of a pharmaceutical compositionfor injection in humans, it has been observed that the leptin amino acidsequence is insoluble at physiologic pH at relatively highconcentrations, such as above about 2 mg active protein/milliliter ofliquid. The poor solubility of leptin under physiological conditionsappears to contribute to the formation of leptin precipitates at theinjection site in a concentration dependent manner when high dosages areadministered in a low pH formulation. Associated with the observedleptin precipitates is an inflammatory response at the injection sitewhich includes a mixed cell infiltrate characterized by the presence ofeosinophils, macrophages and giant cells.

[0007] To date, there have been no reports of stable preparations ofhuman OB protein at concentrations of at least about 2 mg/ml atphysiologic pH, and further, no reports of stable concentrations ofactive human OB protein at least about 50 mg/ml or above. Thedevelopment of leptin forms which would allow for high dosage withoutthe aforementioned problems would be of great benefit. It is thereforeone object of the present invention to provide improved forms of leptinby way of site-specific chemical modification of the protein.

[0008] There are several methods of chemical modification of usefultherapeutic proteins which have been reported. One such method,succinylation, involves the conjugation of one or more succinyl moietiesto a biologically active protein. Classic approaches to succinylationtraditionally employ alkaline reaction conditions with very largeexcesses of succinic anhydride. The resultant succinyl-proteinconjugates are typically modified at multiple sites, often show alteredtertiary and quaternary structures, and occasionally are inactivated.The properties of various succinylated proteins are described inHolcenberg et al., J. Biol. Chem, 250:4165-4170 (1975), and WO 88/01511(and references cited therein), published Mar. 10, 1988. Importantly,none of the cited references describe methods wherein the biologicallyactive protein is monosuccinylated exclusively at the N-terminus of theprotein, and wherein the resultant composition exhibits improvedsolubility and improved injection site toxicity's.

[0009] Diethylenetriaminepentaacetic acid anhydride (DTPA) andethylenediaminetetraacetic acid dianhydride (hereinafter referred to asEDTA²) have classically been used to introduce metal chelation sitesinto proteins for the purpose of radiolabeling. Similar tosuccinylation, modification with DTPA and/or EDTA² typically occurs atmultiple sites throughout the molecule and changes the charge andisoelectric point of the modified protein. To date, there have been noreports of DTPA- and/or EDTA²-protein monomers and dimers which exhibitimproved solubility and improved injection site toxicity's.

SUMMARY OF THE INVENTION

[0010] The present invention relates to substantially homogenouspreparations of chemically modified proteins, e.g. leptin, and methodstherefor. Unexpectedly, site-specific chemical modification of leptindemonstrated advantages in bioavailibility and biocompatibility whichare not seen in other leptin species. Importantly, the methods describedherein are broadly applicable to other proteins (or analogs thereof), aswell as leptin. Thus, as described below in more detail, the presentinvention has a number of aspects relating to chemically modifyingproteins (or analogs thereof) as well as specific modifications ofspecific proteins.

[0011] In one aspect, the present invention relates to a substantiallyhomogenous preparation of mono-succinylated leptin (or analog thereof)and related methods. Importantly, the method described results in a highyield of monosuccinylated protein which is modified exclusively at theN-terminus, thereby providing processing advantages as compared to otherspecies. And, despite the modest N-terminal modification, themonosubstituted succinyl-leptin unexpectedly demonstrated: 1) asubstantial improvement in solubility; 2) preservation of secondarystructure, in vitro receptor binding activity and in vivo bioefficacy;and 3) amelioration of the severe injection site reactions observed withadministration of high concentrations of unmodified leptin.

[0012] In another aspect, the present invention relates to substantiallyhomogenous preparations of DTPA-leptin monomers and dimers and relatedmethods. When reacted with leptin at neutral pH and a low stoichiometricexcess of DTPA:protein, this reagent unexpectedly forms a singlecrosslink between the N-termini of two leptin molecules in high yield.When the monosubstituted DTPA-leptin monomer and dimer are isolated,both show substantially increased solubility's relative to theunmodified protein. Both forms also demonstrate preservation of in vitroreceptor binding activity and in vivo bioefficacy. Significantly, thedimeric form of monosubstituted DTPA-leptin did not precipitate wheninjected at high concentration in PBS and demonstrated strongimprovement in the injection site reactions over those observed with theunmodified leptin.

[0013] In yet another aspect, the present invention relates tosubstantially homogenous preparations of EDTA dianhydride (EDTA²)-leptinmonomers and dimers and related methods. Similar to DTPA in structure,EDTA² crosslinks leptin efficiently through the N-terminus when allowedto react at neutral pH in a substoichiometric excess. The isolatedEDTA²-leptin dimer demonstrates dramatically enhanced solubilityrelative to unmodified leptin and maintains full in vitro receptorbinding activity and in vivo bioactivity. Furthermore, the EDTA²-leptinconjugate did not precipitate at the injection site when dosed at highconcentration in PBS and demonstrated substantial improvement in theadverse injection site reactions observed with the unmodified leptin.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 is a chromatogram of a anion exchange chromatographyseparation of succinylated leptin. Absorbance at 280 nm is plotted vs.elution volume in mL. The monosuccinylated leptin peak is marked by (*).

[0015]FIG. 2 is a pH 3-7 IEF-PAGE gel depicting unmodified leptin (lane2), succinylated leptin (lane 3), DTPA modified leptin dimer (lane 4)and EDTA² modified leptin dimer (lane 5). Lanes 1 and 6 are isoelectricpoint markers.

[0016]FIG. 3 is a chromatogram of a size exclusion chromatographyseparation of DTPA crosslinked leptin monomer and dimer. Absorbance at280 nm is plotted vs. elution volume in mL. The dimeric form ofmonosubstituted DTPA-leptin is marked by (*).

[0017]FIG. 4 is a 4-20% SDS-PAGE gel depicting unmodified leptin (lane2), succinylated leptin (lane 3), DTPA modified leptin dimer (lane 4)and EDTA² modified leptin dimer (lane 5). Lanes 1 and 6 are molecularweight markers.

[0018]FIG. 5 is a chromatogram of a size exclusion chromatographyseparation of EDTA² crosslinked leptin monomer and dimer. Absorbance at280 nm is plotted vs. elution volume in mL. The dimeric form ofmonosubstituted EDTA²-leptin is marked by (*).

[0019]FIG. 6 is a reverse phase HPLC chromatogram of Lys-C digestsshowing retention time shifts resulting from chemical modifications ofthe N-terminal peptide (M1-K6) by succinic anhydride.

[0020]FIG. 7 is a reverse phase HPLC chromatogram of Lys-C digestsshowing retention time shifts resulting from chemical modifications ofthe N-terminal peptide (M1-K6) by DTPA.

[0021]FIG. 8 is a reverse phase HPLC chromatogram of Lys-C digestsshowing retention time shifts resulting from chemical modifications ofthe N-terminal peptide (M1-K6) by EDTA².

[0022]FIG. 9 depicts Far-UV CD spectra of unmodified native leptin andmonosuccinylated leptin. Both samples are at 0.25 mg/mL in phosphatebuffered saline at ambient temperature.

[0023]FIG. 10 is a graph depicting in vitro receptor binding ofunmodified leptin (-♦-), succinylated-leptin (- -), DTPA-leptin dimer(-Δ-) or EDTA²-leptin dimer (--) by displacement of radiolabeled humanleptin from immobilized human leptin receptor. Ligand concentration(ng/mL) is plotted versus % ligand bound.

[0024]FIG. 11 is a graph depicting weight loss in mice that had beentreated with either unmodified leptin (-♦-), succinylated-leptin (- -),DTPA-leptin dimer (-Δ-) or DTPA-leptin monomer (-x-). Mice were doseddaily at 10 mg/kg delivered at 2 mg/mL in PBS. Time (days) is plottedversus % weight loss.

[0025]FIG. 12 is a graph depicting weight loss in mice that had beentreated with either 20 mg/mL unmodified leptin (-Δ-), 2 mg/mL unmodifiedleptin (-♦-), 20 mg/mL EDTA²-leptin dimer (- -) or 2 mg/mL EDTA²-leptindimer (-x-). Mice were dosed daily at 100 mg/kg delivered at 20 mg/mL or10 mg/kg delivered at 2 mg/mL in PBS (unmodified leptin dosed at 100mg/kg and 20 mg/mL was formulated in pH 4.0, acetate buffer due to itspoor solubility in PBS). Time (days) is plotted versus % weight loss.

DETAILED DESCRIPTION

[0026] The present invention relates to substantially homogenouspreparations of chemically modified proteins, and methods therefor.“Substantially homogenous” as used herein means that the only chemicallymodified proteins observed are those having one “modifier” (e.g., DTPA,EDTA², succinyl) moiety. The preparation may contain unreacted (i.e.,lacking modifier moiety) protein. As ascertained by peptide mapping andN-terminal sequencing, one example below provides for a preparationwhich is at least 90% modified protein, and at most 10% unmodifiedprotein. Preferably, the chemically modified material is at least 95% ofthe preparation (as in the working example below) and most preferably,the chemically modified material is 99% of the preparation or more. Thechemically modified material has biological activity. The present“substantially homogenous” monosuccinylated leptin, DTPA-leptin, andEDTA²-leptin preparations provided herein are those which are homogenousenough to display the advantages of a homogenous preparation, e.g., easein clinical application in predictability of lot to lotpharmacokinetics.

[0027] As used herein, biologically active agents refers to recombinantor naturally occurring proteins, whether human or animal, useful forprophylactic, therapeutic or diagnostic application. The biologicallyactive agent can be natural, synthetic, semi-synthetic or derivativesthereof. In addition, biologically active agents of the presentinvention can be perceptible. A wide range of biologically active agentsare contemplated. These include but are not limited to hormones,cytokines, hematopoietic factors, growth factors, antiobesity factors,trophic factors, anti-inflammatory factors, and enzymes (see also U.S.Pat. No. 4,695,463 for additional examples of useful biologically activeagents). One skilled in the art will readily be able to adapt a desiredbiologically active agent to the compositions of present invention.

[0028] Such proteins would include but are not limited to interferons(see, U.S. Pat. Nos. 5,372,808, 5,541,293, 4,897,471, and 4,695,623hereby incorporated by reference including drawings), interleukins (see,U.S. Pat. No. 5,075,222, hereby incorporated by reference includingdrawings), erythropoietins (see, U.S. Pat. Nos. 4,703,008, 5,441,868,5,618,698, 5,547,933, and 5,621,080 hereby incorporated by referenceincluding drawings), granulocyte-colony stimulating factors (see, U.S.Pat. Nos. 4,810,643, 4,999,291, 5,581,476, 5,582,823, and PCTPublication No. 94/17185, hereby incorporated by reference includingdrawings), stem cell factor (PCT Publication Nos. 91/05795, 92/17505 and95/17206, hereby incorporated by reference including drawings), andleptin (OB protein) (see PCT publication Nos. 96/40912, 96/05309,97/00128, 97/01010 and 97/06816 hereby incorporated by referenceincluding figures). PCT publication No. WO 96/05309, published Feb. 22,1996, entitled, “Modulators of Body Weight, Corresponding Nucleic Acidsand Proteins, and Diagnostic and Therapeutic Uses Thereof” fully setsforth OB protein and related compositions and methods, and is hereinincorporated by reference. An amino acid sequence for human OB proteinis set forth at WO 96/05309 Seq. ID Nos. 4 and 6 (at pages 172 and 174of that publication), and the first amino acid residue of the matureprotein is at position 22 and is a valine residue. The mature protein is146 residues (or 145 if the glutamine at position 49 is absent, Seq. IDNo. 4).

[0029] In addition, biologically active agents can also include but arenot limited to insulin, gastrin, prolactin, adrenocorticotropic hormone(ACTH), thyroid stimulating hormone (TSH), luteinizing hormone (LH),follicle stimulating hormone (FSH), human chorionic gonadotropin (HCG),motilin, interferons (alpha, beta, gamma), interleukins (IL-1 to IL-12),tumor necrosis factor (TNF), tumor necrosis factor-binding protein(TNF-bp), brain derived neurotrophic factor (BDNF), glial derivedneurotrophic factor (GDNF), neurotrophic factor 3 (NT3), fibroblastgrowth factors (FGF), neurotrophic growth factor (NGF), bone growthfactors such as osteoprotegerin (OPG), insulin-like growth factors(IGFs), macrophage colony stimulating factor (M-CSF), granulocytemacrophage colony stimulating factor (GM-CSF), megakaryocyte derivedgrowth factor (MGDF), keratinocyte growth factor (KGF), thrombopoietin,platelet-derived growth factor (PGDF), colony simulating growth factors(CSFs), bone morphogenetic protein (BMP), superoxide dismutase (SOD),tissue plasminogen activator (TPA), urokinase, streptokinase andkallikrein. The term proteins, as used herein, includes peptides,polypeptides, consensus molecules, analogs, derivatives or combinationsthereof.

[0030] In general, comprehended by the invention are pharmaceuticalcompositions comprising effective amounts of chemically modifiedprotein, or derivative products, together with pharmaceuticallyacceptable diluents, preservatives, solubilizers, emulsifiers, adjuvantsand/or carriers needed for administration. (See PCT 97/01331 herebyincorporated by reference.) The optimal pharmaceutical formulation for adesired biologically active agent will be determined by one skilled inthe art depending upon the route of administration and desired dosage.Exemplary pharmaceutical compositions are disclosed in Remington'sPharmaceutical Sciences (Mack Publishing Co., 18th Ed., Easton, Pa.,pgs. 1435-1712 (1990)). The pharmaceutical compositions of the presentinvention may be administered by oral and non-oral preparations (e.g.,intramuscular, subcutaneous, transdermal, visceral, IV (intravenous), IP(intraperitoneal), intraarticular, placement in the ear, ICV(intracerebralventricular), IP (intraperitoneal), intraarterial,intrathecal, intracapsular, intraorbital, injectable, pulmonary, nasal,rectal, and uterine-transmucosal preparations).

[0031] Therapeutic uses of the compositions of the present inventiondepend on the biologically active agent used. One skilled in the artwill readily be able to adapt a desired biologically active agent to thepresent invention for its intended therapeutic uses. Therapeutic usesfor such agents are set forth in greater detail in the followingpublications hereby incorporated by reference including drawings.Therapeutic uses include but are not limited to uses for proteins likeinterferons (see, U.S. Pat. Nos. 5,372,808, 5,541,293, herebyincorporated by reference including drawings), interleukins (see, U.S.Pat. No. 5,075,222, hereby incorporated by reference includingdrawings), erythropoietins (see, U.S. Pat. Nos. 4,703,008, 5,441,868,5,618,698, 5,547,933, and 5,621,080 hereby incorporated by referenceincluding drawings), granulocyte-colony stimulating factors (see, U.S.Pat. Nos. 4,999,291, 5,581,476, 5,582,823, 4,810,643 and PCT PublicationNo. 94/17185, hereby incorporated by reference including drawings), stemcell factor (PCT Publication Nos. 91/05795, 92/17505 and 95/17206,hereby incorporated by reference including drawings), and the OB protein(see PCT publication Nos. 96/40912, 96/05309, 97/00128, 97/01010 and97/06816 hereby incorporated by reference including figures). Inaddition, the present compositions may also be used for manufacture ofone or more medicaments for treatment or amelioration of the conditionsthe biologically active agent is intended to treat.

[0032] The principal embodiment of the method for making thesubstantially homogenous preparation of monosuccinylated proteincomprises: (a) reacting a protein with 3-7 fold molar excess of succinicanhydride; (b) stirring the reaction mixture 2-16 hours at 4° C.; (c)dialyzing said mixture against 20 mM Tris-HCl, pH 7.2; and (d) isolatingsaid monosuccinylated protein. Optionally, the method can comprise, justafter step (b), the steps of: adding solid hydroxylamine to said mixturewhile maintaining the pH above 6.5 until said hydroxylamine iscompletely dissolved, followed by elevating the pH to 8.5 using 5 NNaOH, followed by stirring said mixture another 1-2 hours at 4° C. Thegeneral process is shown schematically in Example 1.

[0033] The principal embodiment of the method for making thesubstantially homogenous preparation of DTPA-protein comprises: (a)reacting a protein with 1-5 fold molar excess of DTPA; (b) stirring thereaction mixture 2-16 hours at 4° C.; (c) dialyzing said mixture against20 mM Tris-HCl, pH 7.2; and (d) isolating said DTPA-protein. The generalprocess is shown schematically in Example 1.

[0034] The principal embodiment of the method for making thesubstantially homogenous preparation of EDTA²-protein comprises: (a)reacting a protein with 0.5-5 fold molar excess of EDTA²; (b) stirringthe reaction mixture 2-16 hours at 4° C.; (c) filtering said reactionmixture; (d) concentrating said reaction mixture; and (e) isolating saidEDTA²-protein. The general process is shown schematically in Example 1.

[0035] The following examples are offered to more fully illustrate theinvention, but are not to be construed as limiting the scope thereof.Example 1 describes the preparation of monosuccinylated leptin,monosubstituted DTPA-leptin monomers and dimers, and EDTA²-leptinmonomers and dimers. Example 2 describes the physiochemicalcharacterization of the modified leptin species prepared in Example 1.Example 3 describes the receptor binding studies performed on themodified leptin species prepared in Example 1. Example 4 describes thesolubility testing performed on the modified leptin species prepared inExample 1. Example 5 describes the in vivo bioactivity studies performedon the modified leptin species prepared in Example 1. Example 6describes the injection site evaluation performed on the modified leptinspecies prepared in Example 1.

EXAMPLE 1

[0036] This example describes the preparation of monosuccinylatedleptin, monosubstituted DTPA-leptin monomers and dimers, andEDTA²-leptin monomers and dimers.

1. Monosuccinylated Leptin

[0037] The protein succinylation method of the present invention can begenerally depicted as follows:

[0038] Recombinant human-methionyl-leptin (rhu-met-leptin) protein(prepared as described in Materials and Methods, infra) at 2-3 mg/mL in20 mM NaHPO₄, pH 7.0, was reacted with 3-7 fold molar excess of solidsuccinic anhydride (Sigma Chemical, St. Louis, Mo.), with a 5 fold molarexcess preferred, and the reaction stirred 2-16 hours at 4° C. Solidhydroxylamine (Sigma Chemical, St. Louis, Mo.) is then added to thereaction while maintaining the pH above 6.5. After the hydroxylamine hasdissolved completely the pH is elevated to 8.5 using 5 N NaOH and thereaction allowed to stir another 1-2 hours at 4° C. (the hydroxylaminestep may be omitted with a small decrease in yield). Finally, thereaction is dialyzed against 20 mM Tris-HCl, pH 7.2.

[0039] The monosuccinylated rhu-met-leptin is isolated by anion exchangechromatography with a High Performance Sepharose Q column (Pharmacia,Piscataway, N.J.) in 20 mM Tris, pH 7.2, with a 0-0.5 M NaCl gradient(see FIG. 1). The product is recognized in the eluant by an isoelectricshift of −0.7 pI units observed with isoelectric focusing (IEF) PAGEusing a 5% polyacrylamide, pH 3-7 gel (Novex, Inc., San Diego, Calif.)(FIG. 2). Final recovery of monosuccinylated rhu-met-leptin is typically45-47%.

2. Monosubstituted DTPA-leptin Monomers and Dimers

[0040] The DTPA modification method of the present invention can begenerally depicted as follows:

[0041] Recombinant human-methionyl-leptin (rhu-met-leptin) protein(prepared as described in Materials and Methods, infra) at 2-3 mg/mL in20 mM NAHPO₄, pH 7.0, was reacted with a 1-5 fold molar excess of solidDTPA (Sigma Chemical, St. Louis, Mo.), with 2-3 fold molar excesspreferred, and the reaction stirred 2-16 hours at 4° C. Finally, thereaction is dialyzed against 20 mM Tris-HCl, pH 7.2. The DTPA modifiedrhu-met-leptin is isolated by anion exchange chromatography with a HighPerformance Sepharose Q column (Pharmacia, Piscataway, N.J.) in 20 mMTris, pH 7.2, with a 0-0.5 M NaCl gradient. Alternatively, monomeric anddimeric forms of monosubstituted DTPA-rhu-met-leptin or rhu-met-leptinare separated by size exclusion chromatography on a Sephacryl 100 column(Pharmacia, Piscataway, N.J.) in PBS (Life Technologies, Grand Island,N.Y.)(see FIG. 3). The products are recognized in the eluant by anisoelectric shift observed with the monomeric DTPA-leptin by isoelectricfocusing (IEF) PAGE using a 5% polyacrylamide, pH 3-7 gel (Novex, Inc.,San Diego, Calif.)(FIG. 2) or the mass increase of a crosslinked dimerobserved with SDS-PAGE using a 4-20% polyacrylamide gel (Novex, Inc.,San Diego, Calif.)(see FIG. 4). Final recovery of DTPA-rhu-met-leptindimer is approximately 30%.

3. Monosubstituted EDTA²-leptin Monomers and Dimers

[0042] The EDTA² modification method of the present invention can begenerally depicted as follows:

[0043] Recombinant human-methionyl-leptin (rhu-met-leptin) protein(prepared as described in Materials and Methods, infra) at 2-3 mg/mL in20 mM NaHPO₄, pH 7.0, was reacted with a 0.5-5 fold molar excess ofEDTA² (Aldrich Chemical Co., Milwaukee, Wis.) either as a solid ordissolved in DMSO, with 0.75 fold molar excess EDTA² in DMSO preferred,and the reaction stirred 2-16 hours at 4° C.

[0044] The reaction is then filtered through a 0.45 micron filter(Nalgene), concentrated by stirred cell over 10 kDa molecular weightcutoff membrane to ˜20 mg/mL and the monomeric and dimeric forms ofmonosubstituted EDTA²-rhu-met-leptin then separated by size exclusionchromatography on a Sephacryl 100 column (Pharmacia, Piscataway, N.J.)equilibrated in PBS (see FIG. 5). Alternatively, the reaction may bepurified by hydrophobic interaction chromatography using a HighPerformance Phenyl-Sepharose column (Pharmacia, Piscataway, N.J.) elutedwith a 0.8-0 M ammonium sulfate gradient in 20 mM NaHPO₄, pH 7.0. Theproducts are recognized in the eluant by an isoelectric shift observedwith the monomeric EDTA²-rhu-met-leptin by isoelectric focusing (IEF)PAGE using a 5% polyacrylamide, pH 3-7 gel (Novex, Inc., San Diego,Calif.) (FIG. 2) or the mass increase of a crosslinked dimer observedwith SDS-PAGE using a 4-20% polyacrylamide gel (Novex, Inc., San Diego,Calif.)(FIG. 4). Final recovery of EDTA²-rhu-met-leptin dimer exceeds50%.

EXAMPLE 2

[0045] This example describes the physiochemical characterization of theleptin conjugates prepared in Example 1. Modification ofsuccinyl-leptin, DTPA-leptin monomers and dimers, and EDTA²-leptinmonomers and dimers was evaluated by a combination of peptide mapping ofLys-C digests on reverse phase HPLC, MALDI-TOF mass spectrometry andpeptide sequencing.

[0046] Lys-C digests of unmodified leptin and the various modifiedleptins were performed by reaction of 100 μg of protein with 4 μg ofendoproteinase Lys-C (Boehringer Mannheim) in 50 mM Tris-HCl, pH 8.5(200 μl) for four hours at room temperature. Peptide maps of the varioussamples were generated by reverse phase HPLC on a 4.6×250 mm, 5 μ C4column (VydaK, Hesperia, Calif.) equilibrated in 0.1% triflouroaceticacid (TFA) with elution over a 0-90% acetonitrile gradient (see FIGS.6-8). As evidenced by the plots depicted in FIGS. 6-8, only theN-terminal peptide (M1-K6) shows any change in retention time as aresult of chemical modification. This result indicates that lysine atposition 6 is unmodified and accessible to Lys-C digestion and suggeststhat the chemical modification occurs at the α-amine of the N-terminus.N-terminal modification is further supported by efforts at N-terminalsequencing which indicate that the N-terminus is blocked (data notshown).

[0047] Mass determinations for succinyl-leptin and DTPA- andEDTA²-leptin dimers were made on a Kompact Maldi IV (Kratos, Ramsey,N.J.) using a 12 pmol sample in a sinapinic acid matrix. Each conjugateindicates a single chemical modification per molecule. TABLE 1 ExpectedMass Linker Mass Measured Mass Conjugate (Da) (Da) (Da) Unmod. leptin16,157 0 16,156 Succinyl-leptin 16,258 101 16,254 DTPA-leptin dimer32,671 357 32,705 EDTA²-leptin dimer 32,570 256 32,509

[0048] In addition to the analysis above, the effects on the secondarystructure of the succinyl-leptin was evaluated using circular dichroismspectroscopy. Far-UV circular dichroism spectra of unmodified andsuccinylated leptin in phosphate buffered saline were collected using a0.05 cm cell in a Jasco J-710 circular dichroism spectrophotometer(Jasco, Tokyo, Japan). The spectra are depicted in FIG. 9 anddemonstrate that the secondary structure of succinylated-leptin ispreserved.

[0049] In sum, the Example 2 data confirms the modification ofsuccinyl-leptin, DTPA-leptin monomers and dimers, and EDTA²-leptinmonomers and dimers at the N-terminus, as well as preservation ofsecondary structure with succinyl-leptin.

EXAMPLE 3

[0050] This example describes the receptor binding studies performed oneach of the leptin conjugates prepared in Example 1. Each of the leptinconjugates prepared in Example 1 was evaluated using an in vitroreceptor binding assay which measures the relative affinity of leptinconjugates based on their ability to displace radiolabeled human leptinfrom a human leptin receptor expressed in immobilized cell membranes. Asevidenced by the FIG. 10 data, the chemically modified isoforms,succinyl-, DTPA-, and EDTA²-leptin each showed relative affinities forhuman leptin receptor equal to the unmodified leptin over the entirerange of ligand binding (˜1-100 ng/mL), with ED₅₀'s of approximately 10ng/mL.

[0051] The Example 3 data thus show that the monosubstitutedsuccinyl-leptin, monosubstituted DTPA-leptin dimer, and EDTA²-leptindimer demonstrate preservation of in vitro receptor binding activity ascompared to unmodified leptin.

EXAMPLE 4

[0052] This example describes the solubility testing performed on eachof the leptin conjugates prepared in Example 1. The leptin conjugateswere dialyzed into PBS then concentrated with CentriPrep concentrators,10 kDa molecular weight cutoff (Amicon) to the point that precipitateswere observed. The sample was clarified by centrifugation and theconjugate protein concentration in the supernatant determined. Thesamples were then kept at room temperature (≈22° C.) for 48 hours and atregular time points centrifuged and the conjugate protein concentrationin the supernatant redetermined. The solubility of the conjugate proteinin PBS is thus defined as the steady state protein concentration at roomtemperature observed in the supernatant after centrifugation (see Table2). TABLE 2 Sample Maximum Solubility in PBS (mg/ml) unmodified leptin3.2 succinyl-leptin 8.4 DTPA-leptin 31.6 EDTA²-leptin 59.9

[0053] The Table 2 data shows that the monosubstituted succinyl-leptin,monosubstituted DTPA-leptin, and monosubstituted EDTA²-leptin havesubstantially improved solubility as compared to unmodified leptin, withthe monosubstituted EDTA²-leptin showing dramatically enhancedsolubility.

EXAMPLE 5

[0054] This example describes the in vivo bioactivity studies performedon the leptin conjugates prepared in Example 1. The described leptinconjugates were tested in both mouse and dog animal models to determinebioefficacy relative to the unmodified leptin. Mice were injected dailyfor 5-7 days with monosubstituted succinyl-leptin, DTPA-leptin dimer,DTPA-leptin monomer and EDTA²-leptin dimer at dosages of 1, 10 and 50mg/kg body weight. Bioefficacy was measured as a percentage weight lossfrom day 0, normalized to the vehicle alone control and compared to theweight loss observed with the unmodified protein. All samples fordosages of 1 and 10 mg/kg were formulated in PBS at 0.2 and 2.0 mg/mlrespectively. Higher dosages were formulated in PBS at 20-50 mg/ml forthe chemically modified forms, however the solubility limits of theunmodified leptin necessitated its formulation at high concentrations ina pH 4 acetate buffer. In addition dogs were injected with 0.05, 0.15and 0.5 mg/kg daily dosages of succinyl-leptin at 5 mg/ml over 28 dayswhile monitoring weight loss followed by a recovery period.

[0055] Bioactivity, as judged by drug induced weight loss in animalmodels, for succinyl leptin was equivalent to the unmodified leptin inboth dogs and mice (FIG. 11). Similarly, both DTPA-leptin monomers anddimers and EDTA²-leptin dimers caused equivalent weight loss in mice ascompared to the unmodified leptin (FIGS. 11 & 12).

[0056] The FIG. 11 & 12 data show that the monosubstitutedsuccinyl-leptin, monosubstituted DTPA-leptin monomers and dimers, andEDTA²-leptin dimer demonstrate preservation of in vivo bioefficacy ascompared to unmodified leptin.

EXAMPLE 6

[0057] This example describes the injection site evaluation performed onthe leptin conjugates prepared in Example 1. Tissue sections from theinjection sites of three mice from each dosing group were examinedhistochemically. Injection site pathology's which were identified andscored were necrosis, suppurative (mixed cell infiltrate composed ofeosinophils and neutrophils), mononuclear cells (macrophages), leptinprecipitates (characterized as either fine ppt. or largedeposits/clumps) and giant cells. Each reaction was scored using thefollowing grading system:

[0058] 0 Normal

[0059] 0.5-1 Minimal change

[0060] 1.5-2 Mild change

[0061] 2.5-3 Moderate change

[0062] 3.5-4 Marked change

[0063] 4.5-5 Massive change

[0064] The averaged sum of the scores for each animal were used todefine an overall biocompatibility score using the following scoringkey: 0-2 Normal 3-5 Minimal  6-10 Mild 11-20 Moderate 21-30 Marked >30Severe

[0065] Although high concentrations of succinyl-leptin were marginallysoluble in PBS at pH 7.0, for the purposes of injection site testing,samples of succinyl-leptin at 20 mg/ml remained soluble in PBS at pH 7.2and at 50 mg/ml in PBS at pH 7.5. Table 3 shows the injection siteevaluation comparing unmodified leptin at 50 mg/mL delivered in pH 4.0,acetate buffer vs. monosubstituted succinyl-leptin at 50 mg/mL in pH7.5, PBS, after 7 days. TABLE 3 Treatment Dose mg/kg Volume mL Necr.Supp. Fine Mono. Large Precip Deposit Giant Cells Acetate Buf 0 20 0 0.51 0 0 1 0 20 0 0 0.5 0 0 0 0 20 0 0.5 1 0 0 0 Unmod. Leptin 50 20 0 3 21 4 1 50 20 0 2.5 2 1 4 2.5 50 20 0 1.5 2 0 1.5 1 PBS Buffer 0 20 0 0.50.5 0 0 0 0 20 0 0.5 0.5 0 0 0 0 20 0 0 0 0 0 0 Succ-leptin 50 20 0 11.5 0 0 0 50 20 0 2 1 0 0.5 0.5 50 20 0 1.5 0.5 0 0 0

[0066] As depicted in Table 3, monosubstituted succinyl-leptin, at highconcentration dosages, showed improvement in every category of injectionsite pathology relative to the unmodified leptin, with the most dramaticimprovement seen with the almost complete elimination of leptinprecipitates and giant cells in the injection sites.

[0067] Table 4 shows the injection site evaluation comparing unmodifiedleptin at 43 mg/mL delivered in pH 4.0, acetate buffer vs.monosubstituted succinyl-leptin at 43 mg/mL in pH 4.0, acetate buffer,after 7 days.

[0068] The Table 4 data shows that, surprisingly, it was also observedthat high concentrations of monosubstituted succinyl-leptin could bedelivered in pH 4, acetate buffer and still demonstrate the dramaticimprovements in injection site reactions observed when monosubstitutedsuccinyl-leptin was delivered in PBS.

[0069] Table 5 shows the injection site evaluation comparing unmodifiedleptin at 20 mg/mL delivered in pH 4.0, acetate buffer vs.monosubstituted DTPA-leptin dimer at 20 mg/mL in PBS, after 7 days.TABLE 5 Treatment Dose mg/kg Volume mL Necr. Supp. Mono. Biocomp. PrecipScore Reaction Acetate Buf 0 80 0 0.25 1 0 3 minimal 0 80 0 0 0.5 0 1normal 0 80 0.25 0.25 1 0 4 minimal Unmod. Leptin 20 80 0 2.5 2.5 2 16moderate 20 80 0.25 3.5 3 2 22 marked 20 80 0.25 3 3 2.5 21 marked PBSBuffer 0 80 0 0 0 0 0 normal 0 80 0 0 0.5 0 1 normal 0 80 0 0 0 0 0normal DTPA-lep dimer 20 80 0.25 1.5 2 0 11 moderate 20 80 0 1 1.5 0 7mild 20 80 0 1.5 2 0 10 mild

[0070] Table 6 shows the injection site evaluation comparing unmodifiedleptin at 20 mg/mL delivered in pH 4.0, acetate buffer vs.monosubstituted EDTA²-leptin dimer at 20 mg/mL in PBS, after 7 days.TABLE 6 Treatment Dose mg/kg Volume mL Necr. Supp. Mono. Biocomp. PrecipScore Reaction Acetate Buf 0 100 0 0.25 1 0 3 minimal 0 100 0 0.5 1 0 4minimal 0 100 0 0.5 1 0 4 minimal Unmod. Leptin 100 100 0 2 3 3 18moderate 100 100 0.5 2 3 3 18 moderate 100 100 0 2 2.5 2 14 moderate PBSBuffer 0 100 0 0 0.5 0 1 normal 0 100 0 0.25 0.25 0 1 normal 0 100 00.25 0.25 0 1 normal EDTA-lep dimer 100 100 0 1.5 2 0 9 mild 100 100 01.5 1.5 0 8 mild 100 100 0.5 2.5 3 0 16 moderate

[0071] As depicted in Tables 5 & 6, DTPA-leptin dimers (Table 5) orEDTA²-leptin dimers (Table 6) can be administered to mice at highconcentration in PBS demonstrating the same improvement in injectionsite pathology as observed with succinyl-leptin. These conjugateshowever, are substantially more soluble in pH 7, PBS and thus providefor a more rugged formulation in this buffer.

[0072] In sum, the Example 6 data shows that the monosubstitutedsuccinyl-leptin, monosubstituted DTPA-leptin monomers and dimers, andEDTA²-leptin monomers and dimers do not precipitate at the injectionsite when dosed at high concentrations, and importantly, demonstratesubstantial improvement in the adverse injection site reactions observedwith the unmodified leptin.

Materials and Methods

[0073] 1. Preparation of Recombinant Human Methionyl-leptin Protein

[0074] The present recombinant human methionyl-leptin (rhu-met-leptin)may be prepared according to the above incorporated-by-reference PCTpublication, WO 96/05309 at pages 151-159. For the present workingexamples, a rhu-met-leptin was used which has (as compared to the aminoacid sequence at page 158) a lysine at position 35 instead of anarginine, and an isoleucine at position 74 instead of an isoleucine.Other recombinant human leptin proteins may be prepared according tomethods known generally in the art of expression of proteins usingrecombinant DNA technology.

[0075] While the present invention has been described in terms ofcertain preferred embodiments, it is understood that variations andmodifications will occur to those skilled in the art. Therefore, it isintended that the appended claims cover all such equivalent variationswhich come within the scope of the invention as claimed.

What is claimed is:
 1. A substantially homogenous preparation of amonosuccinylated protein, wherein said monosuccinylated protein ismodified exclusively at the N-terminus.
 2. A substantially homogenouspreparation of an ethylenediaminetetraacetic acid dianhydride(EDTA²)-protein monomer.
 3. A substantially homogenous preparation of anEDTA dianhydride (EDTA²)-protein dimer.
 4. A substantially homogenouspreparation of a diethylenetriaminepentaacetic acid anhydride(DTPA)-protein monomer.
 5. A substantially homogenous preparation of aDTPA-protein dimer.
 6. The substantially homogenous preparationaccording to any of claims 1-5, wherein said protein is leptin, or ananalog thereof.
 7. A monosuccinylated protein produced by a processcomprising the steps of: (a) reacting a protein with 3-7 fold molarexcess of succinic anhydride to form a reaction mixture; (b) stirringsaid reaction mixture 2-16 hours at 4° C.; (c) dialyzing said reactionmixture against 20 mM Tris-HCl, pH 7.2; and (d) isolating saidmonosuccinylated protein from said reaction mixture, wherein saidmonosuccinylated protein is modified exclusively at the N-terminus. 8.The monosuccinylated protein of claim 7 , wherein said protein isleptin, or an analog thereof.
 9. The monosuccinylated protein of claim 7, wherein said protein is leptin, or an analog thereof.
 10. ADTPA-protein produced by a process comprising the steps of: (a) reactinga protein with 1-5 fold molar excess of DTPA to form a reaction mixture;(b) stirring said reaction mixture 2-16 hours at 4° C.; (c) dialyzingsaid reaction mixture against 20 mM Tris-HCl, pH 7.2; and (d) isolatingsaid DTPA-protein from said reaction mixture.
 11. The DTPA-protein ofclaim 10 , wherein said protein is leptin, or an analog thereof.
 12. AEDTA²-protein produced by a process comprising the steps of: (a)reacting a protein with 0.5-5 fold molar excess of EDTA² to form areaction mixture; (b) stirring said reaction mixture 2-16 hours at 4°C.; (c) filtering said reaction mixture; (d) concentrating said reactionmixture; and (e) isolating said EDTA²-protein from said reactionmixture.
 13. The EDTA-protein of claim 12 , wherein said protein isleptin, or an analog thereof.
 14. A method of making a substantiallyhomogenous preparation of a monosuccinylated protein comprising thesteps of: (a) reacting a protein with 3-7 fold molar excess of succinicanhydride to form a reaction mixture; (b) stirring said reaction mixture2-16 hours at 4° C.; (c) elevating the pH of said reaction mixture to8.5 using 5 N NaOH; (d) stirring said reaction mixture another 1-2 hoursat 4° C.; (e) dialyzing said reaction mixture against 20 mM Tris-HCl, pH7.2; and (f) isolating said monosuccinylated protein from said reactionmixture.
 15. The method according to claim 14 further comprising, justafter step (b), the steps of: 1) adding solid hydroxylamine to saidmixture while maintaining the pH above 6.5 until said hydroxylamine iscompletely dissolved; 2) elevating the pH to 8.5 using 5 N NaOH; and 3)stirring said mixture another 1-2 hours at 4° C.
 16. A method of makinga substantially homogenous preparation of a DTPA-protein comprising thesteps of: (a) reacting a protein with 1-5 fold molar excess of DTPA toform a reaction mixture; (b) stirring said reaction mixture 2-16 hoursat 4° C.; (c) dialyzing said reaction mixture against 20 mM Tris-HCl, pH7.2; and (d) isolating said DTPA-protein from said reaction mixture. 17.A method of making a substantially homogenous preparation of aEDTA²-protein comprising the steps of: (a) reacting a protein with 0.5-5fold molar excess of EDTA² to form a reaction mixture; (b) stirring saidreaction mixture 2-16 hours at 4° C.; (c) filtering said reactionmixture; (d) concentrating said reaction mixture; and (e) isolating saidEDTA²-protein from said reaction mixture.
 18. A pharmaceuticalcomposition comprising a monosuccinylated protein.
 19. A pharmaceuticalcomposition comprising EDTA dianhydride (EDTA)-protein monomer.
 20. Apharmaceutical composition comprising EDTA dianhydride (EDTA²)-proteindimer.
 21. A pharmaceutical composition comprising a DTPA-proteinmonomer.
 22. A pharmaceutical composition comprising a DTPA-proteindimer.
 23. A pharmaceutical composition according to any of claims 17-21wherein said protein is leptin, or an analog thereof.
 24. Apharmaceutical composition according to any of claims 18-22 wherein saidprotein is G-CSF, or an analog thereof.